We are using the single genome sequencing (SGS) technology we developed in the previous period to analyze and understand the accumulation of genetic variation in gag/pol and env in a number of different patient groups, including chronically infected patients, both nave and on therapy, as well as in primary and early HIV infection (in collaboration with Drs. Joseph Margolick, Eric Daar, and Shyam Kottilil). The goals of this study are to understand the nature of the forces (mutation, selection, drift, recombination) that mold the genetic diversity of virus populations in vivo. This analysis is being further extended by developing two new sequencing applications: 1) We are applying the SGS technique to analyze the proviral DNA content of cells from patients in parallel with analysis of plasma-derived HIV, as a means of understanding the distribution of the virus population in the body, as well as the potential for recombination. 2) In collaboration with Dr. Mellors, we are developing a new assay (multiple genome sequencing, or MGS) as a potentially efficient way to detect resistance mutations in virus populations at low frequency.We are obtaining a more comprehensive picture of HIV genetic variation on replication in vivo in the presence or absence of drug resistance. We are using the SGS approach to look at resistance more directly, asking, for example, (in collaboration with Dr. Mellors) whether the presence of low-frequency resistance mutations in patients switching from a failed therapy is predictive of subsequent failure. We are extending the portion of the HIV genome we are sequencing to detect novel mutations associated with RT inhibitor resistance in the C-terminal region of RT (in collaboration with Dr. Vinay Pathak). We are also investigating genetic diversity of the gag p6 gene, a viral gene product that binds to host protein TSG101 in cholesterol-laden rafts and is essential for proper virus budding. Following initial observations we made on the inconsistent effects of pravastatin on HIV viral RNA levels in vivo (cross-sectional study in collaboration with Dr. Peter Sklar), we are studying whether gag p6 genetic diversity and specific TSG101 genotypes are associated with viral RNA levels or response to statin therapy in a new randomized multi-center clinical protocol developed during the review period (NIH protocol 01-I-0197, in collaboration with Drs. Eric Freed, Mary Carrington, Anuradha Ganesan, and Peter Sklar).